Lmna mutant genes and uses thereof

Abstract

This application provides LMNA Mutated genes and their applications belong to the field of biomedical technology. Humans LMNA The nucleotide sequence of the mutated gene is shown in SEQ ID NO.1. LMNA Mutant genes can be used for screening dilated cardiomyopathy and atrioventricular block, and for constructing non-human animal models of dilated cardiomyopathy and atrioventricular block. This is beneficial for exploring the pathophysiological processes of dilated cardiomyopathy and atrioventricular block, as well as for discovering therapeutic targets and drugs for dilated cardiomyopathy and atrioventricular block.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically involving... LMNA Mutant genes and their applications. Background Technology

[0002] Dilated cardiomyopathy (DCM) is a complex and progressively worsening heart disease characterized by enlarged cardiac chambers, thinning of the ventricular walls, and impaired cardiac systolic function. These pathological changes directly lead to severe impairment of the heart's pumping function, often causing various arrhythmias and conduction system disorders. As the disease progresses, the patient's cardiac function continues to deteriorate, potentially developing into refractory heart failure with a high mortality rate, placing a heavy burden on patients' families and society. Currently, the clinical treatment of DCM still faces significant challenges, and there is a lack of effective methods for a complete cure.

[0003] In the genetic etiology of DCM, the lamin A / C gene ( LMNA ) plays a key role. (By) LMNA Cardiac lesions caused by mutations are characterized by early involvement of the cardiac conduction system, including conduction disorders such as atrioventricular block and bundle branch block, coexisting with a cardiomyopathy phenotype. The inheritance pattern is autosomal dominant. Compared with other types of DCM, LMNA Related cardiomyopathy has a higher degree of malignancy and a faster disease progression rate. Patients have a significantly increased risk of malignant arrhythmias and sudden death, and respond poorly to conventional drug treatment, seriously threatening their long-term survival and quality of life. LMNA Genes exhibit high mutational heterogeneity, with significant differences in clinical phenotypes resulting from mutations at different sites. Some mutations may only lead to isolated cardiomyopathy, while others can manifest as multisystem diseases with severe conduction disturbances as the initial or primary feature, posing a serious challenge to accurate diagnosis and personalized treatment. Many unknowns remain. LMNA The inability to definitively determine the pathogenicity of gene mutation sites is a major bottleneck in achieving precision medicine. Therefore, further discovery of new... LMNA Gene mutation sites are of great significance for studying the specific mechanisms by which they cause heart disease, for the diagnosis of heart disease or to assist in clinical judgment, and for the discovery of drug targets for intervention. Summary of the Invention

[0004] In view of this, this application provides a LMNA Mutant genes and their applications.

[0005] Firstly, this application provides a human LMNA Mutant genes, the human LMNAThe nucleotide sequence of the mutated gene is shown in SEQ ID NO.1.

[0006] Secondly, this application provides a human LMNA Mutant protein, the human LMNA The amino acid sequence of the mutant protein is shown in SEQ ID NO.2.

[0007] Thirdly, this application provides a non-human animal LMNA Mutated genes, the non-human animals LMNA The coding sequence of the mutated gene is similar to that of the human wild type. LMNA The sequence GCTTGGCG is inserted between the two bases corresponding to positions 142 and 143 of the gene's coding sequence, wherein the human wild-type... LMNA The coding sequence of the gene is shown in SEQ ID NO.3.

[0008] Fourthly, this application provides a method for detecting the human described in the first aspect. LMNA Mutated genes, detection of the human described in the second aspect LMNA Mutant proteins or detection of non-human animals as described in the third aspect LMNA Application of reagents containing mutated genes in the preparation of kits for detecting dilated cardiomyopathy and / or atrioventricular block.

[0009] Fifthly, this application provides a kit for detecting dilated cardiomyopathy and / or atrioventricular block, including the detection of the method described in the first aspect. LMNA The reagents and detection methods for mutated genes described in the second aspect LMNA Reagents or detection methods for mutant proteins in non-human animals as described in the third aspect LMNA Reagents for mutated genes.

[0010] Sixthly, this application provides a targeting vector, including a vector encoding a non-human animal as the target. LMNA Mutant gene or fragment thereof, which encodes a target non-human animal LMNA The mutated gene or its fragment is similar to the human wild type. LMNA A GCTTGGCG insertion is made between the bases corresponding to positions 142 and 143 of the gene's coding sequence, in the human wild-type... LMNA The coding sequence of the gene is shown in SEQ ID NO.3.

[0011] Seventhly, this application provides the targeting vector described in the sixth aspect for constructing non-human animal models of dilated cardiomyopathy, constructing non-human animal models of atrioventricular block, or constructing non-human animal models of... LMNA Application of mutant genes in non-human animal models.

[0012] Eighthly, this application provides a method for constructing a non-human animal model with dilated cardiomyopathy, a non-human animal model with atrioventricular block, or a non-human animal model with... LMNA Methods for constructing non-human animal models of mutated genes include: constructing a non-human animal model of dilated cardiomyopathy, constructing a non-human animal model of atrioventricular block, or constructing a non-human animal model of mutated genes using the targeting vector described in the sixth aspect. LMNA Non-human animal models with mutated genes; or gene editing technology used to modify non-human animals. LMNA The coding sequence is similar to that of the human wild type. LMNA Inserting GCTTGGCG between the bases corresponding to positions 142 and 143 of the gene's coding sequence yields non-human animal models of dilated cardiomyopathy, atrioventricular block, or other conditions. LMNA Non-human animal models of mutated genes, specifically the human wild-type. LMNA The coding sequence of the gene is shown in SEQ ID NO.3.

[0013] Ninthly, this application provides non-human animal models of dilated cardiomyopathy, atrioventricular block, or other conditions prepared by the method described in aspect eight. LMNA Application of non-human animal models of mutated genes in screening drugs for the prevention or treatment of dilated cardiomyopathy and / or atrioventricular block.

[0014] Tenthly, this application provides non-human animal models of dilated cardiomyopathy, atrioventricular block, or other conditions prepared by the method described in aspect eight. LMNA Application of non-human animal models of mutated genes in the preparation of products for evaluating the therapeutic effects on dilated cardiomyopathy and / or atrioventricular block.

[0015] This application provides a novel human study related to dilated cardiomyopathy and atrioventricular block. LMNA Mutated genes, and correspondingly provide information about the human... LMNA Methods for detecting mutated genes enable timely disease screening and treatment intervention; and based on this, [the following is also mentioned:] human... LMNA Mutated genes, resulting in non-human animals LMNA The study also provides mutated genes, targeting vectors for constructing non-human animal models, and methods for constructing such models. The resulting non-human animal models of dilated cardiomyopathy and atrioventricular block can simulate the pathogenesis and more objectively reflect the disease process. LMNA Mutated genes lead to the pathological features of diseases. In-depth exploration of the pathogenesis of gene mutations is of great significance for further discovering therapeutic targets and drugs that can be intervened in dilated cardiomyopathy and atrioventricular block. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. The specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0017] Figure 1 This is a diagram of mouse gene sequencing results.

[0018] Figure 2 Electrocardiograms of mice in each group.

[0019] Figure 3 Electrophoretic thermal mapping of mice in each group.

[0020] Figure 4 Atrial and ventricular field potential maps of mice in each group.

[0021] Figure 5 The graph shows the results of atrioventricular conduction time for each group of mice.

[0022] Figure 6 Electrocardiogram of mice in the KI group.

[0023] Figure 7 Echocardiograms of mice in each group.

[0024] Figure 8 The graph shows the left ventricular ejection fraction results for each group of mice.

[0025] Figure 9 The graph shows the results of the left ventricular short axis shortening rate for each group of mice.

[0026] Figure 10 The graph shows the results of the left ventricular end-systolic diameter in each group of mice.

[0027] Figure 11 The graph shows the results of the left ventricular end-diastolic diameter in mice of each group.

[0028] Figure 12 Images of isolated hearts of mice in each group at 30 weeks.

[0029] Figure 13 The image shows the staining results of heart sections from mice in each group at 30 weeks. Detailed Implementation

[0030] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0031] This application provides a human LMNA The mutated gene, whose nucleotide sequence is shown in SEQ ID NO.1. This human... LMNA The mutated gene is associated with dilated cardiomyopathy and atrioventricular block, and can be used to construct non-human animal models of dilated cardiomyopathy and atrioventricular block. It is of great significance for the pathogenesis, detection, intervention and treatment of dilated cardiomyopathy and atrioventricular block.

[0032] The human beings provided in this application LMNA The nucleotide sequence of the mutated gene was obtained from the human wild-type... LMNA The sequence GCTTGGCG was obtained by inserting the sequence between bases 350 and 351 of the transcript sequence of the gene, resulting in the human wild-type. LMNA The transcript number of the gene is NM_170707.4. The human [gene] provided in this application... LMNA The nucleotide sequence of the mutated gene was obtained from the human wild-type... LMNA The human wild-type gene was obtained by inserting GCTTGGCG between the 142nd and 143rd bases of its coding sequence. LMNA The coding sequence of the gene is shown in SEQ ID NO.3. "Wild-type" is a commonly used term in this field; a wild-type gene / amino acid sequence / nucleotide sequence refers to a mutated, original gene / amino acid sequence / nucleotide sequence. The transcript sequence includes an upstream untranslated region, a coding sequence region, and a downstream untranslated region. In the insertion sequence GCTTGGCG, T stands for thymine, C for cytosine, and G for guanine.

[0033] This application provides a human LMNA The mutant protein, whose amino acid sequence is shown in SEQ ID NO.2, is a human mutant protein. LMNA The amino acid sequence corresponding to the mutated gene is shown in SEQ ID NO.2. This human... LMNA The mutant protein is associated with dilated cardiomyopathy and atrioventricular block, and can be used to construct non-human animal models of dilated cardiomyopathy and atrioventricular block. It is of great significance for the pathogenesis, detection, intervention and treatment of dilated cardiomyopathy and atrioventricular block.

[0034] The human beings provided in this application LMNA Mutant genes, humans LMNAThe mutant protein, compared to transcript number NM_170707.4, has the following base changes: c.350_351insGCTTGGCG, and the amino acid change: p.Val49LeufsTer50. The human [protein / protein] provided in this application... LMNA Mutant genes compared to human wild-type LMNA The base changes in the gene's coding sequence are c.142_143insGCTTGGCG.

[0035] This application provides a non-human animal LMNA Mutated genes, in non-human animals LMNA The coding sequence of the mutated gene is similar to that of the human wild type. LMNA The sequence GCTTGGCG is inserted between the two bases corresponding to positions 142 and 143 of the gene's coding sequence. In the human wild-type... ​ The gene coding sequence is shown in SEQ ID NO.3. This non-human animal's... ​ Mutated genes can be used to construct mutation models in non-human animals, which is beneficial for research on dilated cardiomyopathy and atrioventricular block. Understandably, by using mutations from non-human animals... ​ The coding sequence is similar to that of wild-type humans. ​ By comparing the coding sequences of genes, the genetic information of non-human animals can be obtained based on the comparison results. ​ The coding sequence is similar to that of wild-type humans. ​ The two base sites corresponding to the 142nd and 143rd bases in the gene's coding sequence have a GCTTGGCG sequence between them.

[0036] This application provides for detecting the aforementioned humans. ​ Mutant genes, humans ​ Mutant proteins or non-human animals ​ Application of reagents containing mutated genes in the preparation of kits for detecting dilated cardiomyopathy and / or atrioventricular block.

[0037] This application provides for the detection of human wild-type... ​ Application of reagents containing the c.142_143insGCTTGGCG variant in the gene (NM_170707.4) coding region in the preparation of kits for the detection of dilated cardiomyopathy and / or atrioventricular block.

[0038] In one embodiment of this application, the detection of atrioventricular block can be the detection of third-degree atrioventricular block.

[0039] This application provides a kit for detecting dilated cardiomyopathy and / or atrioventricular block, including the detection of the above-mentioned human... ​ Reagents for detecting mutated genes in humans ​Reagents or detection methods for mutant proteins in the aforementioned non-human animals. ​ A reagent for detecting mutated genes. This kit can be used to screen subjects who have or are suspected of having dilated cardiomyopathy and / or atrioventricular block.

[0040] Subjects can be humans or non-human animals, and the type of biological sample is not particularly limited, as long as a sample reflecting the presence of mutations in the biological sample can be extracted from it. For example, the biological sample can be at least one selected from blood, skin, or subcutaneous tissue, such as peripheral blood. The sample can be any nucleic acid sample that can reflect the presence of mutations in the biological sample; for example, it can be whole-genome DNA directly extracted from the biological sample, or a portion of the whole genome containing the coding sequence of a pathogenic gene, or total RNA extracted from the biological sample, or mRNA extracted from the biological sample. Detection of dilated cardiomyopathy can include screening for the risk of dilated cardiomyopathy; detection of atrioventricular block can include screening for the risk of atrioventricular block. When the subject is human, the reagents in the kit can detect mutations in the subject's biological sample. ​ Whether the gene coding sequence contains the insertion sequence GCTTGGCG between bases 142 and 143, or whether the reagents in this kit can detect the presence of this sequence in the subject's biological sample. ​ The transcript sequence is checked for the c.350_351insGCTTGGCG mutation. When the subject is a non-human animal, the reagents in the kit can detect the presence of this mutation in the subject's biological sample. ​ The gene coding sequence is similar to that of the human wild type. ​ Does the sequence GCTTGGCG exist between the two bases corresponding to the 142nd and 143rd bases in the coding sequence of the gene?

[0041] In one embodiment of this application, the reagent can be a reagent for detecting coding genes based on nucleic acid detection technology. Specifically, the reagent can be, but is not limited to, DNA polymerases, primers, probes, reporter markers (such as fluorescent markers) involved in nucleic acid sequencing.

[0042] In one embodiment of this application, the reagent includes at least one of a primer set and a probe. Exemplarily, the primer set sequences are shown in SEQ ID NO.4 and SEQ ID NO.5, and can be used for mouse... ​ The test detects whether a gene contains a corresponding mutation. For example, the primer sets shown in SEQ ID NO.6 and SEQ ID NO.7 can be used to detect human... ​ The test detects whether there are corresponding mutations in the genes.

[0043] In one embodiment of this application, the reagent can be a reagent for detecting a target protein based on protein detection technology. Specifically, the reagent can be, but is not limited to, selected from specific antibodies, etc.

[0044] This application provides a method for detecting dilated cardiomyopathy in humans, including obtaining a human biological sample and detecting [the following] in the human biological sample. ​ Does the coding sequence of the gene contain the insertion sequence GCTTGGCG between bases 142 and 143? ​ The gene transcript sequence is examined for the c.350_351insGCTTGGCG mutation. If the human biological sample contains this mutation, dilated cardiomyopathy is confirmed.

[0045] This application provides a method for detecting atrioventricular block in humans, including obtaining a human biological sample and detecting [the following] in the human biological sample. ​ Does the gene coding sequence contain the insertion sequence GCTTGGCG between bases 142 and 143? ​ The gene transcript sequence is checked for the c.350_351insGCTTGGCG mutation. If the human biological sample contains this mutation, atrioventricular block is confirmed.

[0046] This application provides a method for detecting dilated cardiomyopathy in non-human animals, including obtaining a non-human animal biological sample and detecting [the following] in the non-human animal biological sample. ​ The gene coding sequence is similar to that of the human wild type. ​ The sequence GCTTGGCG is inserted between the two bases corresponding to positions 142 and 143 of the gene's coding sequence. If the above mutation is present in a non-human animal biological sample, dilated cardiomyopathy is confirmed.

[0047] This application provides a method for detecting atrioventricular block in non-human animals, including obtaining a non-human animal biological sample and detecting [the presence of certain substances] in the non-human animal biological sample. ​ The gene coding sequence is similar to that of the human wild type. ​ The sequence GCTTGGCG is inserted between the two bases corresponding to positions 142 and 143 of the gene's coding sequence. If the above mutation is present in a non-human animal biological sample, atrioventricular block is confirmed.

[0048] In one embodiment of this application, nucleic acid detection technology or protein detection technology can be used to detect substances in biological samples. ​ Does the gene have the above-mentioned mutations?

[0049] This application provides a method for treating dilated cardiomyopathy, including obtaining human biological samples or non-human animal biological samples; detecting in the human biological sample...​ The gene coding sequence has an insertion sequence GCTTGGCG or between bases 142 and 143. ​ Gene transcript sequences with the c.350_351insGCTTGGCG mutation, or in non-human animal biological samples ​ The gene coding sequence is similar to that of the human wild type. ​ The sequence GCTTGGCG is inserted between the two bases corresponding to positions 142 and 143 of the gene's coding sequence; therapeutic agents are administered. Specifically, when the biological sample... ​ When a gene exhibits the aforementioned mutations, it confirms dilated cardiomyopathy, and therapeutic agents can be used for treatment. Alternatively, other detection methods, such as physiological indicators of the subject, can be used to determine which therapeutic agent to administer. In one embodiment of this application, the therapeutic agent is selected from anti-dilated cardiomyopathy drugs. In one embodiment of this application, the therapeutic agent can be a β-blocker, such as propranolol or metoprolol.

[0050] This application provides a method for treating atrioventricular block, including obtaining a human biological sample or a non-human animal biological sample; detecting in the human biological sample... ​ The gene coding sequence has an insertion sequence GCTTGGCG or between bases 142 and 143. ​ Gene transcript sequences with the c.350_351insGCTTGGCG mutation, or in non-human animal biological samples ​ The gene coding sequence is similar to that of the human wild type. ​ The sequence GCTTGGCG is inserted between the two bases corresponding to positions 142 and 143 of the gene's coding sequence; therapeutic agents are administered. Specifically, when the biological sample... ​ When a gene has the above-mentioned mutation, it is confirmed that there is atrioventricular block, and a therapeutic agent can be used for treatment; other detection methods, such as the physiological indicators of the subject, can also be used to determine which therapeutic agent to administer.

[0051] This application provides a carrier, the carrier comprising the aforementioned human... ​ Mutant genes or those of the aforementioned non-human animals ​ Mutant gene.

[0052] This application provides a host cell, the host cell comprising the aforementioned human... ​ Mutant genes or those of the aforementioned non-human animals ​ Mutant gene or the aforementioned vector.

[0053] This application provides the aforementioned human... ​ Mutated genes, the aforementioned human ​ Mutant proteins, and the aforementioned non-human animals ​The application of the mutated gene, the aforementioned vector, or the aforementioned host cell in constructing cell models and animal models of dilated cardiomyopathy and / or atrioventricular block. The animal models in this application are non-human animal models.

[0054] This application provides a targeting vector, comprising encoding a target non-human animal. ​ Mutant genes or fragments thereof, encoding target non-human animals ​ The mutated gene or its fragment is similar to the human wild type. ​ The gene's coding sequence contains the insertion of GCTTGGCG between the bases corresponding to positions 142 and 143. This results in the human wild-type gene. ​ The gene coding sequence is shown in SEQ ID NO.3. In other words, the targeting vector includes the coding sequence for non-human animals. ​ The nucleotide sequence or fragment of a mutated gene can be used to replace the corresponding gene sequence in the genome of a target non-human animal, thereby enabling the target non-human animal to... ​ Humans in the encoded sequence ​ Inserting GCTTGGCG between the bases corresponding to the 142nd and 143rd bases in the gene coding sequence is beneficial for constructing animal models of dilated cardiomyopathy and atrioventricular block, thereby facilitating research on dilated cardiomyopathy and atrioventricular block.

[0055] In one embodiment of this application, the targeting vector further comprises homologous sequences upstream and downstream of the target non-human animal gene target site and components of the vector itself. In one embodiment of this application, the targeting vector further comprises positive and negative selection markers, which is beneficial for subsequent screening. In one embodiment of this application, the targeting vector may include a plasmid vector.

[0056] This application provides information on the application of the aforementioned targeting vector in constructing non-human animal models of dilated cardiomyopathy, constructing non-human animal models of atrioventricular block, or constructing non-human animal models of... ​ Application of mutant genes in non-human animal models.

[0057] Different non-human animals ​ Gene coding sequences have high homology, which can identify genes found in the human body. ​ Gene mutation sites bind to different animals ​ In the gene sequence, different animals ​ The gene sequence corresponding to humans ​ Insertion mutations are performed at gene mutation sites to construct targeting vectors and corresponding non-human animal model mutants for medical research. For example, mice... ​The transcript number of the gene is ENSMUST00000029699.13 (https: / / genome.ucsc.edu / cgi-bin / hgGene?hgg_gene=ENSMUST00000029699.13&hgg_type=knownGene&db=mm39). Sequence alignment revealed that the mouse... ​ Bases 391 and 392 of the gene transcript sequence correspond to the human wild type. ​ Bases 350 and 351 of the transcript sequence, mouse ​ The 142nd and 143rd bases in the gene coding sequence correspond to the wild-type human. ​ The 142nd and 143rd bases of the gene's coding sequence; therefore, gene editing technology can be used to modify mice... ​ Inserting GCTTGGCG between the 142nd and 143rd bases of the gene coding sequence can generate mouse models of dilated cardiomyopathy and atrioventricular block.

[0058] This application provides a method for constructing a non-human animal model with dilated cardiomyopathy or for constructing a non-human animal model with dilated cardiomyopathy. ​ Methods for constructing non-human animal models of mutated genes include: constructing a non-human animal model of dilated cardiomyopathy using the aforementioned targeting vector, or constructing a non-human animal model of... ​ Non-human animal models with mutated genes; or gene editing technology used to modify non-human animals. ​ The coding sequence is similar to that of the human wild type. ​ Inserting GCTTGGCG between the bases corresponding to positions 142 and 143 of the gene's coding sequence yields a non-human animal model of dilated cardiomyopathy or a model of... ​ Non-human animal models of mutated genes, human wild type ​ The coding sequence of the gene is shown in SEQ ID NO.3. The above-mentioned targeting vectors or gene editing technologies can be used to modify non-human animals... ​ The coding sequence corresponds to the human wild type. ​ By inserting GCTTGGCG between the bases corresponding to positions 142 and 143 of the gene's coding sequence, a non-human animal model of dilated cardiomyopathy was obtained. ​ Non-human animal models of mutated genes are beneficial for medical research on dilated cardiomyopathy.

[0059] In one embodiment of this application, the gene editing technology is selected from at least one of the following gene editing methods: homologous recombination-based gene targeting technology, zinc finger nuclease technology, transcription activator-like effector nucleases (TALENs) technology, zinc finger nucleases (ENs) technology, CRISPR / Cas9 technology, CRISPR / Cas12a technology, CRISPR / Cas12i technology, prime editing technology, base editing technology, two-base editing technology, single-stranded oligonucleotide (ssODN) or double-stranded DNA template repair technology, and viral vector-mediated integration. This application can use a myocardial-specific transgenic expression system (such as Myh6-Cre, Cx40-Cre) to achieve specific mutation expression in the heart or conduction system. This application can use a chromosome humanization strategy to introduce humanized mutants. ​ The sequence is used to simulate the process of human disease. This application is applicable to various genetic engineering methods such as embryo injection, sperm editing, and in vitro fertilization.

[0060] In one embodiment of this application, the non-human animal or non-human animal model can be a mammal, such as at least one of mice, rats, cattle, Xenopus laevis, chickens, rabbits, dogs, chimpanzees, zebrafish, and pigs.

[0061] This application provides a non-human animal model with dilated cardiomyopathy, a non-human animal model with atrioventricular block, or a non-human animal model with... ​ Non-human animal models of mutated genes, among which ​ The coding sequence of the gene is similar to that of the human wild type. ​ GCTTGGCG was inserted between the two bases corresponding to the 142nd and 143rd bases in the gene coding sequence.

[0062] According to domestic guidelines, the diagnostic criteria for dilated cardiomyopathy are: ① left ventricular end-diastolic diameter (LVEDd) > 5.0 cm (female) and LVEDd > 5.5 cm (male) (or greater than 117% of the predicted value based on age and body surface area, i.e., 2 times the predicted value SD + 5%); ② left ventricular ejection fraction < 45% and left ventricular fractional shortening < 25%; ③ exclusion of hypertension, valvular heart disease, congenital heart disease, or ischemic heart disease at the time of onset. In this application, the following conditions in non-human animal models are considered to indicate dilated cardiomyopathy: increased heart / body weight compared to wild-type controls in the same litter, echocardiography showing decreased left ventricular ejection fraction, decreased left ventricular fractional shortening, increased left ventricular systolic / diastolic diameter, enlarged heart, and HE staining of cardiac sections showing enlarged cardiac chambers.

[0063] According to international guidelines, atrioventricular block is classified into first-degree (prolonged PR interval), second-degree (type I Wenckebach phenomenon, type II PR interval fixed with dropout), and third-degree (complete atrioventricular dissociation) atrioventricular block based on the degree of conduction impairment as assessed by electrocardiogram. In this application, atrioventricular block is considered to be present in non-human animal models if the following conditions are confirmed by electrocardiogram recording: partial or complete interruption of atrioventricular electrical impulse conduction, with electrocardiographic characteristics similar to those in humans; or prolonged atrioventricular conduction time as measured by animal cardiac mapping experiments.

[0064] This application provides non-human animal models of dilated cardiomyopathy, non-human animal models of atrioventricular block, or other similar models. ​ Application of non-human animal models of mutated genes in screening drugs for the prevention or treatment of dilated cardiomyopathy and / or atrioventricular block.

[0065] Specifically, non-human animal models with dilated cardiomyopathy or with ​ Application of non-human animal models with mutated genes in screening drugs for the prevention or treatment of dilated cardiomyopathy; non-human animal models with atrioventricular block or with ​ Application of non-human animal models of mutated genes in screening drugs for the prevention or treatment of atrioventricular block.

[0066] This application provides non-human animal models of dilated cardiomyopathy, non-human animal models of atrioventricular block, or other similar models. ​ Application of non-human animal models of mutated genes in the preparation of products for evaluating the therapeutic effects on dilated cardiomyopathy and / or atrioventricular block.

[0067] Specifically, non-human animal models with dilated cardiomyopathy or with ​ Application of non-human animal models with mutated genes in the preparation of products for evaluating the therapeutic effects on dilated cardiomyopathy; non-human animal models with atrioventricular block or with ​ Application of non-human animal models of mutated genes in the preparation of products for evaluating the therapeutic effects of atrioventricular block. These products may be, but are not limited to, drugs.

[0068] Non-human animal models with dilated cardiomyopathy, non-human animal models with atrioventricular block, or those with ​ Non-human animal models of mutated genes can simulate the pathogenesis of human hereditary DCM or atrioventricular block in vitro, reflecting the disease more objectively. ​ The pathological characteristics of hereditary DCM or atrioventricular block caused by gene mutations are helpful for exploring the pathogenesis of hereditary DCM or atrioventricular block caused by gene mutations, and are of great significance for further discovering drug targets that can be intervened in hereditary DCM or atrioventricular block.

[0069] This application provides a device for detecting dilated cardiomyopathy and / or atrioventricular block, comprising:

[0070] The input module is used to receive the nucleotide sequence of the subject.

[0071] The processing module is used to determine if the subject's nucleotide sequence matches that of a human wild-type human. ​ Compared to the gene coding sequence, GCTTGGCG is inserted between the bases corresponding to positions 142 and 143 to generate a first processing result. This first processing result is used to indicate that the subject has dilated cardiomyopathy and / or atrioventricular block, and is of human wild type. ​ The gene coding sequence is shown in SEQ ID NO.3;

[0072] The output module is used to output the first processing result.

[0073] In one embodiment of this application, the device further includes a pre-storage module for storing human wild-type... ​ Gene coding sequence.

[0074] In one embodiment of this application, the input module is further configured to receive input human wild-type ​ Gene coding sequence.

[0075] In one embodiment of this application, the processing module is further configured to, if it is determined that the nucleotide sequence of the subject is similar to that of a human wild-type... ​ There is no insertion sequence between the 142nd and 143rd bases of the gene's coding sequence, generating a second processing result. This second processing result is used to indicate that the subject does not have dilated cardiomyopathy or atrioventricular block. The output module is also used to output the second processing result.

[0076] The following specific examples further illustrate the effectiveness of the technical solution in this application.

[0077] This application can use an in vitro model as an alternative, such as a knockout wild-type model edited with CRISPR / Prime Editing. ​ Later, mutant cell lines were added back, or AAV9 vectors were used to deliver mutant expression. ​ In vivo and in vitro models of the myocardial system can prove ​ The above mutations affect myocardial structure, electrocardiographic conduction, and myocardial contractile function.

[0078] This application is not limited to telemetry electrocardiography and echocardiography for the detection of atrioventricular block and dilated cardiomyopathy. For the atrioventricular block phenotype: This application employs an implantable telemetry system and ex vivo electrophysiological mapping, which are the gold standard methods for assessing cardiac electrophysiological function. Alternatives may include: surface electrocardiography (suitable for preliminary, non-invasive screening), transesophageal electrophysiological examination, or the use of in vivo / ex vivo electrophysiological recording systems from other manufacturers. These methods can be used as verification methods as long as they can reliably detect abnormalities such as prolonged atrioventricular conduction time and first / second / third-degree atrioventricular block. For the dilated cardiomyopathy phenotype: This invention uses echocardiography to assess cardiac structure and function. Cardiac magnetic resonance imaging (MRI) is an important alternative, providing more accurate information on cardiac volume, ventricular wall thickness, and fibrosis, and is another authoritative means of verifying ventricular dilation and heart failure. In addition, performing cardiac histopathological analysis at the experimental endpoint (such as heart weight / body weight ratio, ventricular cavity diameter measurement, cardiomyocyte cross-sectional area and fibrosis staining) is also an effective alternative method to directly confirm the dilated cardiomyopathy phenotype morphologically.

[0079] Using CRISPR / Cas9 gene editing technology, the gene is injected via microinjection. ​ The donor DNA and gene editing components encoding the c.142_143insGCTTGGCG mutation were introduced into C57BL / 6J mouse zygotes, and the specific mutation was precisely knocked into the mouse genome using homology-directed repair mechanisms. ​ By identifying the corresponding sites in the gene coding sequence, a heterozygous gene knock-in (KI) mouse model with a clearly defined genetic background can be bred. ​ c.142 _143insGCTTGGCG / + The primer sets involved are shown in SEQ ID NO.4 and SEQ ID NO.5. Genomic DNA was extracted from mouse tail tissue, amplified by PCR, and sequenced by Sanger sequencing to identify the genotype, ensuring accurate insertion and stable inheritance of the mutant sequence. The sequencing primers are shown in SEQ ID NO.4. ​ The image shows the mouse gene sequencing results. Unmutated wild-type (WT) mice are designated as the WT group. Compared to the WT group, the homozygous (Ho) group and the heterozygous (He) group... ​ Gene mutation occurs, in ​ The sequence GCTTGGCG was successfully inserted between the 142nd and 143rd bases of the gene coding sequence (e.g., ​ (The red line indicated by the middle arrow). Therefore, the method described above successfully constructed a device carrying... ​Homozygous (Ho) and heterozygous (He) mouse models of the mutated gene were established. All homozygous mice died within 3 weeks of birth. Subsequent phenotypic observations were mainly conducted using wild-type and heterozygous mice, with the heterozygous mice also serving as either the He or KI group mice in subsequent experiments.

[0080] Telemetry devices were subcutaneously implanted in mice in the WT and He groups at 6 weeks of age, and the electrocardiograms of the mice were observed 1-2 weeks later using the DSI fully implantable telemetry system. ​ The electrocardiograms of mice in each group are shown, with the horizontal axis representing time (seconds) and the vertical axis representing voltage (mV). It can be seen that the He mice began to show an atrioventricular block phenotype from week 10 onwards. ​ (The point indicated by the middle arrow).

[0081] Simultaneously, electrophysiological mapping (MappingLab) experiments were performed on mice in the WT and KI groups. Specifically, after isolating and perfusing the mouse heart, two 32-bit electrophysiological mapping pens were placed in the right atrium (RA) and left ventricle (LV) of the mouse heart, respectively, and data were measured 15 minutes after perfusion. ​ Electrophoretic conduction heat maps of mice in each group. ​ The atrial and ventricular field potential maps of mice in each group are shown. ​ The graph shows the atrioventricular conduction time results for each group of mice. The value p < 0.05 indicates that the atrioventricular conduction time in the KI group mice was significantly longer than that in the WT group mice. ​ The images show electrocardiograms (ECGs) of mice in the KI group. Using an ECG element from MappingLab, third-degree atrioventricular block was observed in the hearts of KI group mice within 25 minutes of ex vivo observation. These experiments demonstrate the successful establishment of an atrioventricular block mouse model.

[0082] High-frequency ultrasound imaging was used to perform non-invasive cardiac structure and function testing on 12-week-old WT and KI mice, and key parameters such as left ventricular ejection fraction, left ventricular fractional shortening, and left ventricular end-systolic and end-diastolic diameters were obtained and analyzed. ​ Echocardiograms of mice in each group. ​ The figures show the results for left ventricular ejection fraction (LVEF%), left ventricular fractional shortening (LVFS%), left ventricular end-systolic diameter (LVIDs), and left ventricular end-diastolic diameter (LVIDd) for each group of mice. ​ Images of isolated hearts of mice in each group at 30 weeks. ​ Images showing the staining results of heart sections from each group of mice at 30 weeks. This indicates that p < 0.05. This indicates that p < 0.01. This indicates p < 0.001. It can be seen that at 12 weeks, the KI group mice showed decreased left ventricular ejection fraction, decreased left ventricular fractional shortening, and increased systolic / diastolic diameter, indicating cardiac enlargement and impaired cardiac function, suggesting a dilated cardiomyopathy phenotype. At 30 weeks, the KI group mice showed increased cardiac size, and longitudinal section hematoxylin-eosin (HE) staining of the heart revealed enlarged cardiac chambers and an enlarged heart. These experiments demonstrate the successful establishment of a mouse model of dilated cardiomyopathy.

[0083] It can be seen that the application provides ​ The mutated gene is associated with dilated cardiomyopathy and atrioventricular block. A non-human animal model with dilated cardiomyopathy and atrioventricular block can be constructed. This animal model successfully reproduces the core clinical phenotype of dilated cardiomyopathy and atrioventricular block in human patients, establishing a direct causal relationship between genotype and phenotype. This provides an irreplaceable in vivo research platform for in-depth analysis of the molecular mechanism of this mutation and lays a key experimental foundation for early diagnosis, risk assessment, and future targeted therapy development for patients with this rare mutation.

[0084] The above description represents the preferred embodiments of this application, but should not be construed as limiting the scope of this application. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this application, and these improvements and modifications are also considered to be within the scope of protection of this application.

Claims

1. Detection of mice LMNA The application of reagents containing mutated genes in the preparation of kits for detecting mouse models of dilated cardiomyopathy and / or atrioventricular block, wherein the mice... LMNA The coding sequence of the mutated gene is similar to that of the human wild type. LMNA The sequence GCTTGGCG is inserted between the two bases corresponding to positions 142 and 143 of the gene's coding sequence, wherein the human wild-type... LMNA The coding sequence of the gene is shown in SEQ ID NO.3, and the mouse's LMNA The transcript number of the gene is ENSMUST00000029699.

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2. A targeting vector, characterized in that, The targeting vector is used to target mice. LMNA The coding sequence is similar to that of the human wild type. LMNA A GCTTGGCG insertion is made between the bases corresponding to positions 142 and 143 of the gene's coding sequence, in the human wild-type... LMNA The coding sequence of the gene is shown in SEQ ID NO.3, and the mouse's LMNA The transcript number of the gene is ENSMUST00000029699.

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3. The targeting vector as described in claim 2 can be used to construct mouse models of dilated cardiomyopathy, mouse models of atrioventricular block, or mouse models of... LMNA Application in mouse models of mutant genes.

4. A method for constructing a mouse model with dilated cardiomyopathy, constructing a mouse model with atrioventricular block, or constructing a mouse model with LMNA a mutant gene, characterized in that, include: The targeting vector of claim 2 is used to construct a mouse model with dilated cardiomyopathy, to construct a mouse model with atrioventricular block, or to construct a mouse model with a mutant gene; or LMNA a mutant gene. Using gene editing technology to modify mice LMNA The coding sequence is similar to that of the human wild type. LMNA Inserting GCTTGGCG between the bases corresponding to positions 142 and 143 of the gene's coding sequence yields mouse models with dilated cardiomyopathy, atrioventricular block, or other mouse models. LMNA A mouse model of the mutant gene, the human wild-type LMNA The coding sequence of the gene is shown in SEQ ID NO.3, and the mouse's LMNA The transcript number of the gene is ENSMUST00000029699.

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5. The use of the mouse model obtained by the method of claim 4 in screening drugs for the prevention or treatment of dilated cardiomyopathy and / or atrioventricular block.

6. The use of the mouse model obtained by the method of claim 4 in the preparation of products for evaluating the therapeutic effects on dilated cardiomyopathy and / or atrioventricular block.

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